Regulatory T cells (Tregs) specified by the Forkhead transcription factor Foxp3 are important to suppress unwanted inflammatory responses in order to maintain immune homeostasis. The gut represents a specific microenvironment for Treg-mediated immune tolerance due to large amounts of antigens derived from food or microbiota, and proinflammatory cytokines produced by various immune cells. The aryl hydrocarbon receptor (Ahr) is an environmental sensor that detects not only xenobiotic ligands such as environmental pollutants (e.g., dioxin) but also physiological compounds generated by host cells, microbiota, and/or diet (e.g., amino acid tryptophan metabolites). The goal of the proposal is to understand the molecular regulation of Tregs by Ahr, a ligand-dependent transcription factor. Our preliminary data suggest that Ahr-expressing Tregs were preferentially enriched in the gut. Using an RNA-Seq approach, we showed that multiple genes that have been previously implicated in Treg cell development, maintenance, and function were perturbed by Ahr deficiency in Tregs. Using a T cell transfer model of colitis, we further showed that Tregs lacking expression of Ahr could not suppress disease in vivo. Using a combination of biochemical, molecular, and genetic approaches, in the proposal we will test the hypothesis that Ahr expression in Tregs plays a key role in gut immune homeostasis by regulating Treg cell development, maintenance, and/or function. Specifically, we will determine 1) the mechanism(s) underlying the enrichment of Ahr-expressing Tregs in the gut; 2) the molecular mechanism(s) by which Ahr regulates expression of the molecules that are involved in Treg development and maintenance; and 3) the in vivo role of Ahr in Tregs during gut inflammation and infection. These experiments will offer an opportunity to elucidate environmental impacts on immune tolerance via the Ahr-mediated pathway. Our study will provide novel cellular and molecular insights into the development and function of Tregs regulated by Ahr in a tissue- and cell-specific manner. Since Ahr is a ligand-dependent transcription factor and its activity can be modulated by small molecules, better understanding of the role of Ahr in Tregs may lead to development of novel therapeutic approaches by modulating Ahr expression and/or function in Tregs to treat autoimmunity and inflammation.